Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus.

The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH(2), where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO(2)) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-electron oxidation proceeds in three consecutive, two-electron steps, through p-hydroxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO) intermediates, with each step requiring one equivalent of O(2) and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo- -AurF) formed by addition of O(2) to the diiron(II/II) enzyme ( -AurF) effects the initial oxidation of Ar-NH(2), generating a mu-(oxo)diiron(III/III) form of the enzyme (mu-oxo- -AurF) and (presumably) Ar-NHOH. Here we show that peroxo- -AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO(2) final product, a four-electron oxidation, and produces -AurF, with which O(2) can combine to regenerate peroxo- -AurF. Thus, conversion of Ar-NHOH to Ar-NO(2) requires only a single equivalent of O(2) and (starting from -AurF or peroxo- -AurF) is fully catalytic in the absence of exogenous reducing equivalents, by contrast to the published stoichiometry. This novel type of four-electron N-oxidation is likely also to occur in the reaction sequences of nitro-installing di-iron amine oxygenases in the biosyntheses of other natural products.

A long-lived, substrate-hydroxylating peroxodiiron(III/III) intermediate in the amine oxygenase, AurF, from Streptomyces thioluteus.

The amine oxygenase AurF from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate (pABA) to p-nitrobenzoate (pNBA). In this work, we have studied the reaction of its reduced Fe(2)(II/II) cofactor with O(2), which results in generation of a peroxo-Fe(2)(III/III) intermediate. In the absence of substrate, this intermediate is unusually stable (t(1/2) = 7 min at 20 degrees C), allowing for its accumulation to almost stoichiometric amounts. Its decay is accelerated approximately 10(5)-fold by the substrate, pABA, implying that it is the complex that effects the two-electron oxidation of the amine to the hydroxylamine. The nearly quantitative conversion of pABA to pNBA by solutions containing an excess of the intermediate suggests that it may also be competent for the two subsequent two-electron oxidations leading to the product.